Work has continued on the transcription and translation of LINE-1 retrotransposons (L1Hs) in human teratocarcinoma cells and on the characterization of the polypeptides encoded by these elements. The aim is to understand the mechanism and regulation of transposition. The DNA sequences near the 5' ends of some L1Hs elements are undermethylated in cell lines synthesizing p40 but are methylated in cells with little or no detectable L1Hs expression, suggesting that methylation state plays a role in L1Hs expression. Translation of L1Hs ORF2, which is separated from ORF1 by 33 base pairs including several in-frame stop codons, was studied in vitro; no ORF1-ORF2 fusion protein was detectable, translation appears to initiate with the first AUG codon in ORF 2, and the absence of ORF1 translation had no effect on ORF2 translation. These results indicated that ORF2 translation is independent of ORF1 translation, in vitro. Experiments to study translation in teratocarcinoma cells upon transient transfection with appropriate DNA constructs lead to a similar conclusion; ORF2 translation is initiated internally within the L1Hs mRNA and is independent of ORF1 translation. Thus, the inhibition of ORF1 translation that results from insertion of a stable stem-loop structure in the 5' UTR, is accompanied by an increase in ORF2 translation. p40, the translation product of L1Hs ORF1 and segments of the polypeptide encoded by ORF2 have been expressed in pRSET expression vectors in E. coli. Large amounts of p40 have been purified from the bacterial cells under denaturing conditions and renatured by controlled dialysis. Homomultimeric complexes of p40 form and appear to involve both disulfide and hydrophobic bonds. Experiments with p40 missing its carboxyl terminal region but retaining the leucine zipper portion of the molecule indicated that the carboxyl terminus facilitates formation of the complexes.